With concerns about greenhouse gas emissions and uncertainty about the supply of oil, renewable biofuels have been gaining increasing attention. Butanol has received increasing interest because it can use renewable biomass as feedstock and is environmentally friendly. Butanol can be produced by anaerobic microorganisms such as Clostridium acetobutylicum and Clostridium beijerinckii in acetone-butanol-ethanol fermentation (ABE fermentation), which was once the second largest industrial fermentation in the world. In a typical ABE fermentation, butyrate and acetate are produced first, and then the culture undergoes a metabolic shift and solvents (butanol, acetone, and ethanol) are formed. In conventional ABE fermentation, the butanol yield is low (<25% w/w), titer is low (<12 g/L) and productivity is low (<0.3 g/L·h). This is largely due to the fact that high concentrations of butanol are toxic to the bacteria that produce the solvent. Other byproducts of ABE fermentation, including acetone, ethanol, acetate and butyrate, also inhibit butanol production by the bacteria. The low reactor productivity, butanol yield, and final butanol concentration make biobutanol from ABE fermentation uneconomical for the fuel market. However, if final butanol concentration could be raised from 12 to 19 g/L, the costs of butanol recovery from the fermentation broth could be cut in half, making ABE fermentation a much more desirable source of butanol.
Since the first oil crisis in the early 1980's, there have been numerous attempts to improve butanol production by metabolically engineering bacteria to have higher butanol tolerance and yet maintain butanol productivity. The problem is that metabolic engineering is limited by available molecular or functional knowledge of bacteria.